Although the molecular mechanism of tamoxifen action in estrogen receptor-"positive" (ER+) breast cancer cells is well studied, there is little information on how tamoxifen may act in normal ER-"poor" human mammary epithelial cells (HMECs). This information is critical for developing breast cancer prevention strategies with defined endpoints. To model p53 loss in the context of tamoxifen chemoprevention, we tested in vitro whether tamoxifen was able to eliminate HMECs immediately after suppression of p53 function. Surprisingly, when p53 was acutely suppressed, the resultant p53(-) HMECs underwent apoptosis when treated with clinically relevant concentrations oftamoxifen, p53(+) controls underwent growth arrest only. Induction of apoptosis correlated with a rapid reduction of Akt-1 phosphorylation, decreased expression of Bcl-2, mitochondrial depolarization, and caspase-activation. The "classic" mechanism of steroid action requires the presence of ER and both transcription and translation. However, there is evidence that estrogen and perhaps antiestrogens may act through "non-classic" signaling pathways. In Preliminary Data, we observed that induction of apoptosis was not induced by 4-OHT or ICI 182,780. Based on Preliminary Data we hypothesize that induction of apoptosis by tamoxifen in p53(-) HMECs may occur via a "non-classic" Akt-regulated pathway. While early passage p53(-) HMECs initially exhibited tamoxifen-sensitivity, resistance rapidly developed and correlated with persistent Akt-phosphorylation, lack of mitochondrial depolarization and loss of expression of the CREB-binding protein, CBP. Observations in our model system predict that if HMECs loose p53 function in the absence of tamoxifen chemoprevention there is a high likelihood that tamoxifen-resistance will subsequently develop. SA I will test l) whether ER modulates sensitivity to tamoxifen-induced apoptosis in early passage p53(-) HMECs utilizing phage display, ER-selective peptide ligand and 2) whether tamoxifen induces apoptosis in p53 (-/-) ER-"poor" mammary cells in vivo. SA II will test whether l) inhibition of Akt phosphorylation in p53(-) HMECs promotes tamoxifen-induced apoptosis and 2) Akt mediates CBP-phosphorylation. SA III will test whether tamoxifen promotes apoptosis in early passage p53(-) HEMCs by altering the ratio of Bcl-2/Bad and test the relationship between Akt phosphorylation and Bcl-2/Bad activity. Studies proposed in SAs will characterize this novel mechanism of tamoxifen-signaling and define potential biologic markers of response. Our long-term goal is to utilize this signal-transduction pathway to model novel combinations of hormonal and non-hormonal agents and then pilot these novel strategies in small-scale prevention trials utilizing markers developed in this proposal to test for response.